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Lecture 3 February 7, 2008

Lecture 3 February 7, 2008. Intracellular signaling by receptor-activated cascades. Cell Structure, Signaling and Differentiation Winter Quarter, 2008 Gerry Weinmaster 390A Basic Science Research Building gweinmaster@mednet.ucla.edu.

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Lecture 3 February 7, 2008

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  1. Lecture 3 February 7, 2008 Intracellular signaling by receptor-activated cascades Cell Structure, Signaling and Differentiation Winter Quarter, 2008 Gerry Weinmaster 390A Basic Science Research Building gweinmaster@mednet.ucla.edu

  2. Many cell surface receptors signal through protein-protein and protein-lipid interactions that are regulated through protein modifications

  3. Phosphorylation/dephosphorylation of proteins provides a major mechanism for signal transduction Proteinkinase ATP ADP Protein P-Protein P Protein phosphatase

  4. 1900 1960 1910 1920 1930 1940 1950 1970 1980 1990 2000 The History of Protein Phosphorylation The History of Protein Phosphorylation Phospho- serine in proteins 1932 Protein kinase activity 1954 Phospho- tyrosine in fly eggs 1964 Src tyrosine kinase 1979 Phospho -protein discovery 1906 Phospho- tyrosine synthesis 1933 Phosph-orylase kinase 1959 cAMP depen-dent PK 1968 Gleevec approved for CML 2001

  5. The discovery of phosphotyrosine and its role in intracellular signaling

  6. HISTORIC MOMENTS IN THE DISCOVERY OF PHOSPHOTYROSINE + Pi Pi 18/9/79 P.SER P.SER “X” P.THR P.TYR P.THR _ | mT acid pH 1.7 pH 1.9 14/6/79 | LT in vivo | mT acid | mT protease | IgH/Src Old Buffer New Buffer

  7. Comparison of 1-D and 2-D phosphoamino acid analysis of phosphorylated Polyoma virus middle T antigen 1-D PAA analysis 2-D PAA analysis Eckhart, Hutchinson and Hunter, Cell 18:925 (1979)

  8. RSV (v-src) transformed cells have increased levels in cellular phosphotyrosine uninfected RSV-transformed Hunter and Sefton, PNAS 77:1311 (1980)

  9. A temperature sensitive strain of RSV showed that tyrosine phosphorylation of cellular proteins was dynamic and dependent on the v-src protein Cells shifted to new temperature for 30 minutes Sefton et al. Cell 20:807 (1980)

  10. Identification of v-Src substrates by 2D gel analysis Alkali-treated Alkali-treated p36 (annexin II) Uninfected chick cells RSV-transformed chick cells Cooper and Hunter, MCB

  11. Substrate P P P P P P P Immune-kinase Assay Adenosine (ATP) + Fc Protein A Bead Intrinsic kinase activity

  12. P P P P P P P P Immune-kinase Assay Adenosine adaptor Adaptor (ATP) + kinase Fc Protein A Bead Associated kinase activity

  13. ? EGFR INSR PDGFR  FLT1 FGFR1 MET TRKA AXL LTK ROR1 TIE HER2 IGF1R FLK1 FGFR2 RON TRKB MER ALK ROR2 TEK PDGFR  HER3 FLT4 FGFR3 TRKC SKY IRR CSF1R HER4 FGFR4 KIT FLK2 LMR1 LMR2 LMR3 Receptor tyrosine kinases are grouped based on specific extracellular structural motifs as well as related kinase sequences CCK4 RYK DDR1 DDR2 RET ROS MuSK RTK106 EphA1 EphA2 EphA3 EphA4 EphA5 EphA6 EphA7 EphA8 EphB1 EphB2 EphB3 EphB4 EphB5 EphB6 LET-23 T01G5.1 T17A3.8 W04G5.6N W04G5.6C Y50D4B-4 ZK938.5 B0198.3 F54F7.5 DAF-2 F59F3.1 F59F3.5 F40G9.13 EGL-15 KIN15 KIN16 TKR-1 C08H9.8 F59F5.3 M01B2.1 R09D1.12 R09D1.13 C16B8.1 F11D5.3 C25F6.4 C16D9.2 CAM-1 T10H9.2 Human RTKs 58 (20 classes) Worm RTKs 29 (11 classes) 11 Unclassified VAB-1 (B0252.1, F11E6.8, F40A3.5, R151.4, T148.1, T22B11.3 Y38H6C.20, C24G6.2A, F08F1.1, F09A5.2, F09G2.1)

  14. Some cell surface receptors use associated non-receptor tyrosine kinases to effect signal transduction through tyrosine phosphorylation

  15. Non-receptor tyrosine kinases have structural motifs that facilitate interactions with cell surface receptors, the plasma membrane, signaling effectors and targets

  16. Model for ligand-induced RTK dimerization and activation of intrinsic kinase activity through transphosphorylation

  17. Raf MEK The PDGF receptor is the major tyrosine phosphoryated protein following treatment of cells with PDGF

  18. Membrane-associated docking proteins interact with activated receptors to provide addition sites of tyrosine phosphorylation for SH2 proteins

  19. SH2 containing proteins bind to phosphotyrosine sites in activated receptors and docking proteins to effect downsteam signal transduction

  20. Receptor tyrosine kinase signaling involves multiple interacting motifs that direct modular protein-protein interactions

  21. PDGF receptor tyrosine phosphorylation allows interactions with multiple downstream effectors and signaling pathways Raf MEK

  22. Ras activation downstream of EGFR occurs via the Grb2 SH2/SH3 adaptor protein Constitutive protein-protein interaction through SH3-proline rich domains EGF-induced protein-protein interaction through SH2-PY interactions

  23. A cascade of kinase activation transmits signals downstream of Ras to MAP kinase MAPKKK MAPKK

  24. The active MAP kinase dimer translocates to the nucleus to induce target genes

  25. RTK phosphorylation allows downstream effectors to bind to membrane lipids and generate second messengers for signaling Raf MEK

  26. PI-3 kinase activity is recruited to the membrane by activated RTKs to generate docking lipids that activate downstream signaling effectors PTEN dephosphorylates PtdIns(3,4,5)P3 to regulate cell growth and survival

  27. How many tyrosine kinases are there? • The finding that v-Src and c-Src phosphorylate tyrosine • provided the first evidence for tyrosine kinase in 1979 • By the end of 1980 four tyrosine kinases were known (Src, Abl, EGF receptor, Fps/Fes) • By the end of 1990 over 50 tyrosine kinases had been • identified in vertebrates and equal numbers of tyrosine • kinases and serine kinases were known, leading to the • prediction that there might be several 100 tyrosine • kinases in a vertebrate genome and a total of over a • 1000 protein kinases • The complete human genome sequence reported in 2001 • reveals that there are 90 tyrosine kinases (all the tyrosine • kinases had been found by other means before the sequence • was completed), out of a total of 518 protein kinases

  28. What is tyrosine phosphorylation used for? 1. Growth factor signaling (and oncogenesis) 2. Cell adhesion, spreading, migration and shape 3. Cell differentiation in development 4. Cell cycle control 5. Gene regulation and transcription 6. Endocytosis and exocytosis 7. Insulin stimulation of glucose uptake 8. Angiogenesis (formation of new blood vessels) 9. Regulation of ion channels in nerve transmission

  29. Therapeutic intervention for receptor tyrosine kinases Gschwind et al. Nature Reviews Cancer 4:361, 2001

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